NF-κB transmits signals from the cell surface to the nucleus. Signaling through cell surface receptors to activate NF-κB and MAP kinases through adaptor molecules is of critical importance to survival and activation of all cells in the body, including those regulating innate and adaptive immunity, including antigen-presenting cells such as dendritic cells (DC). As such, NF-κB is a key signaling component in autoimmunity and an attractive target for autoimmune disease therapy.
NF-κB Function
Five NF-κB proteins p50, p52, c-Rel, p65/RelA and RelB are present in mammals. All share a rel homology domain (RHD) that mediates DNA-binding, dimerization and nuclear translocation. p50 and p52 homodimers are transcriptionally inactive, but have the capacity to bind DNA. In contrast, c-rel or Rel A are able to bind DNA and p50 or p52 are able to bind DNA and mediate transcriptional activation. As a result, the availability of subunits and affinity determines the NF-κB composition of the cell (Hoffmann A, Baltimore D: Circuitry of nuclear factor kappaB signaling. Immunol. Rev. (2006) 210:171-186).
In unstimulated cells, NF-κB is present as an inactive form in the cytoplasm bound to inhibitory proteins or IκBs, including IκBα, IκBβ, IκBε, IκBγ, IκBNS, Bcl-3, p100 and p105 (Ghosh S, M: Missing pieces in the NF-kappaB puzzle. Cell (2002) 109 Suppl:S81-96). These proteins contain ankyrin repeats consisting of two tightly packed helices followed by a loop and a tight hairpin turn, which facilitate binding to NF-κB dimers. The NLS region of NF-κB enables dimer nuclear import. IκBβ masks the NLS, preventing nuclear import of dimers. In contrast, IκBα only masks the NLS of p65 and not p50. Nuclear retention is normally prevented by the presence of a nuclear export sequence in IκBα. If this NF-κB export sequence is blocked, RelA/p50 complexes are retained in the nucleus (Huang Tt, Kudo N, Yoshida M, Miyamoto S: A nuclear export signal in the N-terminal regulatory domain of IkappaBalpha controls cytoplasmic localization of inactive NF-kappaB/IkappaBalpha complexes. Proc. Natl. Acad. Sci. U.S.A. (2000) 97(3):1014-1019).
A variety of receptor-ligand pairs activate NF-κB, including TLR/pathogen signals, inflammatory receptors (TNFR/TNF and IL-1R/IL-1), T cell (CD40/CD40L, TCR/MHC peptide) and B cell signals (BAFFR/BAFF, BCR/Ag) and differentiation signals such as lymphotoxin/LTβ and RANK/RANKL. Signaling these pathways leads to activation of serine/threonine kinase IκB kinase (IKK) (Yamamoto Y, Gaynor R B: IkappaB kinases: key regulators of the NF-kappaB pathway. Trends Biochem. Sci. (2004) 29(2):72-79). IKK phosphorylates IκB which is recognized by a specific ubiquitin ligase complex, b-TrCP-SCF. Ubiquitinated IκB is degraded by the 26S proteasome, leading to release of NF-κB, nuclear import and transcriptional activation. The IKK complex consists of 3 subunits including IKKα (IKK1), IKKβ (IKK2) and the associated non-catalytic regulatory subunit IKKγ/NF-κB essential modulator (NEMO). IKK may be activated through phosphorylation by mitogen activated protein kinase kinase kinase (MAPKKK) or NF-κB inducing kinase (NIK), leading to subsequent autophosphorylation of the IKK complex and full activity. IKKβ and NEMO deficient mice have impaired NF-κB activation in response to cytokine and TLR activation, particularly activation of RelA/p50. In contrast, IKKα has a particular role in activation of RelB/p52 complexes and histone phosphorylation to enhance NF-κB DNA binding.
The differential role of IKKα and IKKβ/NEMO in activating distinct NF-κB subunits has led to the classification of the NF-κB pathway into the classical and alternate pathways (referred to collectively herein as “the NF-κB pathway”) (Xiao G, Rabson A B, Young W, Qing G, Qu Z: Alternative pathways of NF-kappaB activation: a double-edged sword in health and disease. Cytokine Growth Factor Rev. (2006) 17(4):281-293). The classical pathway is activated by TLR and pro-inflammatory cytokines, leading to IKKβ and NEMO-dependent phosphorylation, degradation of IκB, and subsequent activation of RelA/p50 heterodimers. In the absence of continual signaling the pathway is rapidly shut down, as a result of reduced IKKβ activity and induction of IκB. In contrast the alternate pathway is activated by signals associated with cell differentiation, including LTβ, CD40L and BAFF. RelB/p52 heterodimers are the predominant NF-κB proteins induced, regulated by p100, the precursor to p52, which contains an IκB domain target site for phosphorylation by IKKα. Signal-specific activation of IKKα results in processing of p100 to p52 and activation of RelB/p52. This pathway is characterized by sustained IKKα and long lasting activation of NF-κB. The alternate pathway appears to be an adaptation of the classical NF-κB pathway for cellular differentiation processes and is important in B cell and DC differentiation and lymphoid organogenesis. NIK appears to be an upstream kinase that activates IKKα. NIK, IKKα and RelB knockout mice share similar defects in lymphoid organogenesis. Importantly, there is some overlap in activation of the classical and alternate pathway; for example, LTβ signals both pathways and resulting target genes are activated (Dejardin E, Droin N M, Delhase M et al.: The lymphotoxin-beta receptor induces different patterns of gene expression via two NF-kappaB pathways. Immunity (2002) 17(4):525-535). LPS, a typical classical pathway activator, also leads to activation of the alternate pathway (Mordmuller B, Krappmann D, Esen M, Wegener E, Scheidereit C: Lymphotoxin and lipopolysaccharide induce NF-kappaB-p52 generation by a co-translational mechanism. EMBO Rep. (2003) 4(1):82-87). This may be essential for efficient differentiation of DC, which up-regulate both NF-κB pathways following antigen encounter and migration into the secondary lymphoid organs. Activation of the alternate pathway ensures that although newly synthesized IκBα inhibits RelA/p50, newly synthesized RelB and processing of p100 to p52 leads to dimer replacement or exchange with RelB/p52 and sustained DC differentiation (Saccani S, Pantano S, Natoli G: Modulation of NF-kappaB activity by exchange of dimers. Mol. Cell. (2003) 11(6):1563-1574).
In immune responses, NF-κB target genes are involved in inflammation, cellular organization and differentiation and proliferation. Tissue macrophages are the major source of NF-κB-induced pro-inflammatory cytokines. NF-κB induced cytokines such as TNFα, IL-1 and IL-6 activate innate responses leading to the release of c-reactive protein (CRP) and complement, and up-regulation of adhesion molecules by local endothelial cells. NF-κB-induced chemokines, including IL-8, MIP-1α, MCP-1, RANTES and eotaxin, and growth factors such as GM-CSF mobilize and redirect myeloid cells to local tissue. The same set of responses as occurs to infection also occurs in inflammatory autoimmune diseases, such as rheumatoid arthritis (RA) and inflammatory bowel disease (IBD).
NF-κB has a role in lymphoid organogenesis through the induction of the chemokines CXC12, CXCL13, CCL21 and CCL19. NF-κB has a role in many stages of B and T cell differentiation (Claudio E, Brown K, Siebenlist U: NF-kappaB guides the survival and differentiation of developing lymphocytes. Cell Death Differ. (2006) 13(5):697-701) including a role for the alternate pathway in NKT cell development and for the classical and alternate pathways in regulatory T cell (Treg) development (Schmidt-Supprian M, Tian J, Grant E P et al.: Differential dependence of CD4+CD25+ regulatory and natural killer-like T cells on signals leading to NF-kappaB activation. Proc. Natl. Acad. Sci. U.S.A. (2004) 101(13):4566-4571; Schmidt-Supprian M, Courtois G, Tian J et al.: Mature T cells depend on signaling through the IKK complex. Immunity (2003) 19(3):377-389; Zheng Y, Vig M, Lyons J, Van Parijs L, Beg A A: Combined deficiency of p50 and cRel in CD4+ T cells reveals an essential requirement for nuclear factor kappaB in regulating mature T cell survival and in vivo function. J. Exp. Med. (2003) 197(7):861-874). c-Rel is also required for efficient IL-2 production by naïve T cells (Banerjee D, Liou H C, Sen R: c-Rel-dependent priming of naive T cells by inflammatory cytokines. Immunity (2005) 23(4):445-458) and T reg are critically dependent on IL-2 for post thymic survival (D'Cruz L M, Klein L: Development and function of agonist-induced CD25+Foxp3+ regulatory T cells in the absence of interleukin 2 signaling. Nat. Immunol. (2005) 6(11):1152-1159; Fontenot J D, Rasmussen J P, Gavin M A, Rudensky A Y: A function for interleukin 2 in Foxp3− expressing regulatory T cells. Nat. Immunol. (2005) 6(11):1142-1151). NF-κB plays an important role in proliferation of lymphocytes as well as non-hematopoetic cells such as synoviocytes, that hyperproliferate in RA. Relevant NF-κB target genes include c-myc, cyclin D1 and anti-apoptotic genes including c-IAP and Bcl-2.
NF-κB in Autoimmune Inflammation
Autoimmune diseases result from a process involving three distinct but related components—a break in self tolerance, development of chronic inflammation in one or several organs, and if ongoing, tissue destruction and its resultant detrimental effects. “Central” tolerance defects are important contributors to spontaneous autoimmune disease. In the fetal and neonatal period, central tolerance is actively maintained in the thymus (Ardavin C: Thymic dendritic cells. Immunol. Today (1997) 18:350-361). During this process, a repertoire of T cells restricted to self-MHC displayed by the thymic cortical epithelium (cTEC) is selected in each individual. In addition, those T cells reactive to self-antigen presented by medullary antigen-presenting cells (APC), which include medullary epithelial cells (mTEC) and medullary dendritic cells (DC), are deleted by negative selection above a threshold of affinity for self antigens presented by those APC (Kappler J W, Roehm N, Marrack P: T cell tolerance by clonal elimination in the thymus. Cell (1987) 49:273-280). Since an affinity threshold applies for central deletion of self-reactive T cells, circulation of low-affinity self-reactive T cells in the periphery is therefore inevitable. Low-level thymic expression and presentation of self-antigens normally expressed by peripheral somatic cells is common. Expression of these antigens is transcriptionally controlled by AIRE, whose expression is in turn controlled by the alternate NF-κB pathway (Anderson M S, Venanzi Es, Klein L et al.: Projection of an immunological self shadow within the thymus by the aire protein. Science (2002) 298(5597):1395-1401). In spontaneous autoimmune models, a variety of defects in the interaction of APC and thymocytes interferes with the normal process of negative selection, thus permitting the release of autoreactive T cells into the periphery, where subsequent environmental events more readily trigger autoimmune disease (Yoshitomi H, Sakaguchi N, Kobayashi K et al.: A role for fungal {beta}-glucans and their receptor Dectin-1 in the induction of autoimmune arthritis in genetically susceptible mice. J. Exp. Med. (2005) 201(6):949-960). Commonly, viral or modified self-antigens, which have not been expressed in the thymus, are presented by peripheral DC to initiate autoimmunity. A number of modified self-antigens have been described in human autoimmune diseases.
Dendritic Cells
It has been proposed that DC are the critical decision making cells in the immune system (Fazekas de St Groth B. The evolution of self-tolerance: a new cell arises to meet the challenge of self-reactivity. Immunol Today. 1998; 19:448-54). Through their role in the generation of central and peripheral tolerance as well as in priming immune responses and stimulation of memory and effector T cells, DC are likely to play essential roles in both the initiation and perpetuation of autoimmunity and autoimmune diseases. However, the understanding of the means by which DC contribute to peripheral tolerance has opened the exciting possibility of harnessing them for antigen-specific immunotherapy of autoimmune diseases and transplantation.
DC are now recognized as essential regulators of both innate and acquired arms of the immune system (Banchereau J, Steinman R M. Dendritic cells and the control of immunity. Nature. 1998 Mar. 19; 392(6673):245-52). They are responsible for the stimulation of naive T lymphocytes, a property that distinguishes them from all other antigen presenting cells (APC). DC are also essential accessory cells in the generation of primary antibody responses (Inaba K, Steinman R M, Van Voorhis W C, Muramatsu S. Dendritic cells are critical accessory cells for thymus-dependent antibody responses in mouse and in man. Proc Natl Acad Sci USA. 1983 October; 80(19):6041-5) and are powerful enhancers of NK cell cytotoxicity (Kitamura H, Iwakabe K, Yahata T, Nishimura S, Ohta A, Ohmi Y, et al. The natural killer T (NKT) cell ligand alpha-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells. J Exp Med. 1999 Apr. 5; 189(7):1121-8). DC are crucial for the initiation of primary immune responses of both helper and cytotoxic T lymphocytes, and thus act as “nature's adjuvant” (Schuler G, Steinman R M. Dendritic cells as adjuvants for immune-mediated resistance to tumors. J Exp Med. 1997 Oct. 20; 186(8):1183-7). Conversely, DC are also involved in the maintenance of tolerance to antigens. DC contribute to thymic central tolerance and shaping of the T cell repertoire by presenting antigens to T cells and deleting those T cells that exhibit strong autoreactivity (Brocker T. Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex class II-expressing dendritic cells. J Exp Med. 1997 Oct. 20; 186(8):1223-32). However, DC also play a role in peripheral tolerance. Here, DC contribute by deletion of autoreactive lymphocytes and expansion of the population of regulatory T cells (Treg). Accordingly, DC offer potential utility in protective and therapeutic strategies for tolerance restoration in autoimmune diseases.
DC precursors from the bone marrow migrate via the bloodstream to peripheral tissues where they reside as immature DC. Immature DC efficiently capture invading pathogens and other particulate and soluble antigens (Ag). After Ag uptake, DC rapidly cross the endothelium of lymphatic vessels and migrate to the draining secondary lymphoid organs. Following the uptake of immunogenic Ag and lymphatic migration, DC undergo a process of maturation, which is characterized by downregulation of the capacity to capture Ag and upregulation of Ag processing and presentation, expression of co-stimulatory molecules and altered dendritic morphology (Steinman R M. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol. 1991; 9:271-96; Cella M, Sallusto F, Lanzavecchia A. Origin, maturation and antigen presenting function of dendritic cells. Curr Opin Immunol. 1997 February; 9(1):10-6; Cella M, Scheidegger D, Palmer-Lehmann K, Lane P, Lanzavecchia A, Alber G. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J Exp Med. 1996 Aug. 1; 184(2):747-52). After presentation of Ag to naive T cells in the T cell area of secondary lymphoid organs, most DC disappear, probably by apoptosis. Thus, under optimal conditions, the same DC sequentially carries out distinct functions such as capture and processing of Ag, Ag presentation to rare, naïve Ag-specific T cells and induction of Ag-specific T cell clonal expansion.
Considering the crucial role of DC in Ag processing and presentation and thus in the regulation of immune reactivity, DC are important directors of immune responsiveness, through the interactions with responding lymphocytes and other accessory cells. Broadly, evidence suggests that under steady state conditions, recruitment of DC precursors into tissues and migration/maturation into secondary lymphoid organs occurs at low rates and may favour tolerance induction. On the other hand, stimulation of immature DC leading to DC maturation and activation may induce a productive immune response (Sallusto F, Lanzavecchia A. Mobilizing dendritic cells for tolerance, priming, and chronic inflammation. J Exp Med. 1999 Feb. 15; 189(4):611-4).
The process of DC maturation can be stimulated by various mechanisms, including pathogen-derived molecules (LPS, DNA, RNA), proinflammatory cytokines (TNFα, IL-1, IL-6), tissue factors such as hyaluronan fragments, migration of DC across endothelial barriers between inflamed tissues and lymphatics, and T cell-derived signals (CD154) (Sparwasser T, Koch E S, Vabulas R M, Heeg K, Lipford G B, Ellwart J W, et al. Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur J. Immunol. 1998 June; 28(6):2045-54; Cella M, Salio M, Sakakibara Y, Langen H, Julkunen I, Lanzavecchia A. Maturation, activation, and protection of dendritic cells induced by double-stranded RNA. J Exp Med. 1999 Mar. 1; 189(5):821-9; De Smedt T, Pajak B, Muraille E, Lespagnard L, Heinen E, De Baetselier P, et al. Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo. J Exp Med. 1996 Oct. 1; 184(4):1413-24). In contrast, anti-inflammatory signals, such as IL-10, TGFβ, prostaglandins, and corticosteroids tend to inhibit maturation (De Smedt T, Van Mechelen M, De Becker G, Urbain J, Leo O, Moser M. Effect of interleukin-10 on dendritic cell maturation and function. Eur J. Immunol. 1997 May; 27(5):1229-35; Geissmann F, Revy P, Regnault A, Lepelletier Y, Dy M, Brousse N, et al. TGF-beta 1 prevents the noncognate maturation of human dendritic Langerhans cells. J. Immunol. 1999 Apr. 15; 162(8):4567-75; de Jong E C, Vieira P L, Kalinski P, Kapsenberg M L. Corticosteroids inhibit the production of inflammatory mediators in immature monocyte-derived DC and induce the development of tolerogenic DC3. J Leukoc Biol. 1999 August; 66(2):201-4). Thus, DC represent an attractive therapeutic target, either to enhance or to attenuate immunity for modulation of disease. To date, ex vivo modulation of DC and exposure to antigen before transfer into an animal or human recipient has been the major approach to achieve protective and therapeutic immunity. This relates in part to complexity of the DC system in the context of a whole person with an immune system disorder, and in part to the difficulty of delivery of specific Ags and immunomodulators to DC in vivo.
Role of NF-κB in Regulating DC Function
The ability of a myeloid DC to induce immunity or tolerance is linked to its maturation state and thus to NF-κB activity (Dhodapkar M V, Steinman R M, Krasovsky J, Munz C, Bhardwaj N. Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med. 2001 Jan. 15; 193(2):233-8; Jonuleit H, Schmitt E, Schuler G, Knop J, Enk A H. Induction of interleukin 10-producing, nonproliferating CD4(+) T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med. 2000 Nov. 6; 192(9):1213-22; Lutz M B, Kukutsch N A, Menges M, Rossner S, Schuler G. Culture of bone marrow cells in GM-CSF plus high doses of lipopolysaccharide generates exclusively immature dendritic cells which induce alloantigen-specific CD4 T cell anergy in vitro. Eur J. Immunol. 2000 April; 30(4):1048-52: Mehling A, Grabbe S, Voskort M, Schwarz T, Luger T A, Beissert S. Mycophenolate mofetil impairs the maturation and function of murine dendritic cells. J. Immunol. 2000 Sep. 1; 165(5):2374-81). Immature DC generated from murine BM induce T cell unresponsiveness in vitro and prolonged cardiac allograft survival (Lutz M B, Suri R M, Niimi M, Ogilvie A L, Kukutsch N A, Rossner S, et al. Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo. Eur J. Immunol. 2000 July; 30(7):1813-22). Various drugs and cytokines, and inhibitors of NF-κB inhibit myeloid DC maturation (de Jong E C, Vieira P L, Kalinski P, Kapsenberg M L. Corticosteroids inhibit the production of inflammatory mediators in immature monocyte-derived DC and induce the development of tolerogenic DC3. J Leukoc Biol. 1999 August; 66(2):201-4; Griffin M D, Lutz W, Phan V A, Bachman L A, McKean D J, Kumar R. Dendritic cell modulation by 1alpha, 25 dihydroxyvitamin D3 and its analogs: a vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vitro and in vivo. Proc Natl Acad Sci USA. 2001 Jun. 5; 98(12):6800-5; Hackstein H, Morelli A E, Larregina A T, Ganster R W, Papworth G D, Logar A J, et al. Aspirin inhibits in vitro maturation and in vivo immunostimulatory function of murine myeloid dendritic cells. J. Immunol. 2001 Jun. 15; 166(12):7053-62; Lee J I, Ganster R W, Geller D A, Burckart G J, Thomson A W, Lu L. Cyclosporine A inhibits the expression of costimulatory molecules on in vitro-generated dendritic cells: association with reduced nuclear translocation of nuclear factor kappa B. Transplantation. 1999 Nov. 15; 68(9):1255-63; Steinbrink K, Wolfi M, Jonuleit H, Knop J, Enk A H. Induction of tolerance by IL-10-treated dendritic cells. J. Immunol. 1997 Nov. 15; 159(10):4772-80; Yoshimura S, Bondeson J, Foxwell B M, Brennan F M, Feldmann M. Effective antigen presentation by dendritic cells is NF-kappaB dependent: coordinate regulation of MHC, co-stimulatory molecules and cytokines. Int Immunol. 2001 May; 13(5):675-83), including corticosteroids, salicylates, mycophenolate mofetil, transforming growth factor (TGF)-β IL-10. DC generated in the presence of these agents alter T cell function in vitro and in vivo, including promotion of allograft survival (Giannoukakis N, Bonham C A, Qian S, Zhou Z, Peng L, Harnaha J, et al. Prolongation of cardiac allograft survival using dendritic cells treated with NF-κB decoy oligodeoxyribonucleotides. Mol. Ther. 2000; 1(5 Pt 1):430-7; Griffin M D, Lutz W, Phan V A, Bachman L A, McKean D J, Kumar R. Dendritic cell modulation by 1alpha, 25 dihydroxyvitamin D3 and its analogs: a vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vitro and in vivo. Proc Natl Acad Sci USA. 2001; 98(12):6800-5; Rea D, van Kooten C, van Meijgaarden K E, Ottenhoff T H, Melief C J, Offringa R. Glucocorticoids transform CD40-triggering of dendritic cells into an alternative activation pathway resulting in antigen-presenting cells that secrete IL-10. Blood. 2000 May 15; 95(10):3162-7; Adorini L, Penna G, Giarratana N, Uskokovic M. Tolerogenic dendritic cells induced by vitamin D receptor ligands enhance regulatory T cells inhibiting allograft rejection and autoimmune diseases. J Cell Biochem. 2003 Feb. 1; 88(2):227-33). NF-κB activity leads to transcription of a number of genes involved in the immune response. In particular, RelB activity is required for myeloid DC differentiation (Burkly L, Hession C, Ogata L, Reilly C, Marconi L A, Olson D, et al. Expression of relB is required for the development of thymic medulla and dendritic cells. Nature. 1995 Feb. 9; 373(6514):531-6; Weih F, Carrasco D, Durham S K, Barton D S, Rizzo C A, Ryseck R P, et al. Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-kappa B/Rel family. Cell. 1995; 80(2):331-40; Wu L, D'Amico A, Winkel K D, Suter M, Lo D, Shortman K. RelB is essential for the development of myeloid-related CD8alpha-dendritic cells but not of lymphoid-related CD8alpha+ dendritic cells. Immunity. 1998 December; 9(6):839-47). RelB regulates DC and B cell APC function through regulation of CD40 and MHC molecule expression (O'Sullivan B J, MacDonald K P, Pettit A R, Thomas R. RelB nuclear translocation regulates B cell MHC molecule, CD40 expression, and antigen-presenting cell function. Proc Natl Acad Sci USA. 2000 Oct. 10; 97(21):11421-6; O'Sullivan B J, Thomas R. CD40 Ligation conditions dendritic cell antigen-presenting function through sustained activation of NF-kappaB. J. Immunol. 2002 Jun. 1; 168(11):5491-8; Martin E, O'Sullivan B, Low P, Thomas R. Antigen-specific suppression of a primed immune response by dendritic cells mediated by regulatory T cells secreting interleukin-10. Immunity. 2003 January; 18(1):155-67). The present inventors have shown that antigen-exposed DC in which RelB function is inhibited lack cell surface CD40, prevent priming of immunity, and suppress previously primed immune responses. While immature DC, which maintain the potential for subsequent activation, were only moderately suppressive of primed immune responses, RelB-deficient DC lacking this potential were much more suppressive (Martin E, O'Sullivan B, Low P, Thomas R. Antigen-specific suppression of a primed immune response by dendritic cells mediated by regulatory T cells secreting interleukin-10. Immunity. 2003 January; 18(1):155-67).
Use of Dendritic Cells for Tolerance
Increasing evidence in humans and rodents strongly suggests that immature or NF-κB-deficient DC may control peripheral tolerance by inducing the differentiation of regulatory T cells (Dhodapkar M V, Steinman R M, Krasovsky J, Munz C, Bhardwaj N. Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med. 2001 Jan. 15; 193(2):233-8; Jonuleit H, Schmitt E, Schuler G, Knop J, Enk A H. Induction of interleukin 10-producing, nonproliferating CD4(+) T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med. 2000 Nov. 6; 192(9):1213-22; Martin E, O'Sullivan B, Low P, Thomas R. Antigen-specific suppression of a primed immune response by dendritic cells mediated by regulatory T cells secreting interleukin-10. Immunity. 2003 January; 18(1):155-67; Roncarolo M G, Levings M K, Traversari C. Differentiation of T regulatory cells by immature dendritic cells. J Exp Med. 2001 Jan. 15; 193(2):F5-9). Thus, repetitive in vitro stimulation of allogeneic human T cells with immature, monocyte-derived dendritic cells leads to the generation of nonproliferating, suppressive, interleukin-10 (IL-10)-producing Treg (Jonuleit H, Schmitt E, Schuler G, Knop J, Enk A H. Induction of interleukin 10-producing, nonproliferating CD4(+) T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med. 2000 Nov. 6; 192(9):1213-22). Dhodapkar et al. injected autologous, monocyte-derived immature DC, pulsed with influenza matrix peptide and keyhole limpet hemocyanin, subcutaneously in two human volunteers. They reported an Ag-specific inhibition of CD8+ T-cell killing activity and the appearance of peptide-specific IL-10-producing T cells, accompanied by a decrease in the number of interferon (IFN)-γ-producing T cells (Dhodapkar M V, Steinman R M, Krasovsky J, Munz C, Bhardwaj N. Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med. 2001 Jan. 15; 193(2):233-8).
CD40 is a key determinant of DC immunogenicity. Inhibition of the RelB transcription factor or of CD40 itself produces regulatory DC that are able to generate IL-10-producing T regulatory cells in vivo (Martin E, O'Sullivan B, Low P, Thomas R. Antigen-specific suppression of a primed immune response by dendritic cells mediated by regulatory T cells secreting interleukin-10. Immunity. 2003 January; 18(1):155-67). Conversely, tumor antigen-specific immunity can be markedly heightened by engineering DC which are able to express CD40 for prolonged periods in vivo (Hanks B A, Jiang J, Singh R A, Song W, Barry M, Huls M H, et al. Re-engineered CD40 receptor enables potent pharmacological activation of dendritic-cell cancer vaccines in vivo. Nat. Med. 2005 February; 11(2):130-7). IL-10 and TGFβ produced by T regulatory cells may contribute to tolerance by limiting expression of MHC class II and co-stimulatory molecules by DC (Jonuleit H, Schmitt E, Schuler G, Knop J, Enk A H. Induction of interleukin 10-producing, nonproliferating CD4(+) T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med. 2000 Nov. 6; 192(9):1213-22; Roncarolo M G, Levings M K, Traversari C. Differentiation of T regulatory cells by immature dendritic cells. J Exp Med. 2001 Jan. 15; 193(2):F5-9).
In conjunction with decreased expression of co-stimulatory molecules, expression of ILT3 and ILT4 may be increased by regulatory DC (Chang C C, Ciubotariu R, Manavalan J S, Yuan J, Colovai A I, Piazza F, et al. Tolerization of dendritic cells by T(S) cells: the crucial role of inhibitory receptors ILT3 and ILT4. Nat. Immunol. 2002 March; 3(3):237-43). These Ig-like inhibitory receptors, related to NK cell killer inhibitory receptors (KIR), are upregulated by the APC as a result of interaction with CD8+CD28− regulatory T cells. These receptors negatively signal monocytes and DC through immunoreceptor tyrosine-based inhibitory motifs (ITIMs) (Colonna M, Nakajima H, Cella M. A family of inhibitory and activating Ig-like receptors that modulate function of lymphoid and myeloid cells. Semin Immunol. 2000; 12(2):121-7; Colonna M, Navarro F, Bellon T, Llano M, Garcia P, Samaridis J, et al. A common inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells. J Exp Med. 1997; 186(11):1809-18; Colonna M, Samaridis J, Cella M, Angman L, Allen R L, O'Callaghan C A, et al. Human myelomonocytic cells express an inhibitory receptor for classical and nonclassical MHC class 1 molecules. J. Immunol. 1998; 160(7):3096-100). CD4+ T cell-induced NFκB activation of APC is reduced in the presence of CD8+CD28− T cells, potentially through this signaling pathway (Chang C C, Ciubotariu R, Manavalan J S, Yuan J, Colovai A I, Piazza F, et al. Tolerization of dendritic cells by T(S) cells: the crucial role of inhibitory receptors ILT3 and ILT4. Nat. Immunol. 2002; 3(3):237-43).
IL-10 is an important cytokine involved in the generation of regulatory T cells by DC. Treatment of DC with IL-10 can convert immature DC into regulatory DC by suppressing NF-κB and therefore arresting maturation. This drives the differentiation of IL-10 producing T regulatory type 1-producing cells in vitro and in vivo (Steinbrink K, Wolfl M, Jonuleit H, Knop J, Enk A H. Induction of tolerance by IL-10-treated dendritic cells. J. Immunol. 1997 Nov. 15; 159(10):4772-80; Steinbrink K, Jonuleit H, Muller G, Schuler G, Knop J, Enk A H. Interleukin-10-treated human dendritic cells induce a melanoma-antigen-specific anergy in CD8(+) T cells resulting in a failure to lyse tumor cells. Blood. 1999 Mar. 1; 93(5):1634-42; Liu L, Rich B E, Inobe J, Chen W, Weiner H L. Induction of Th2 cell differentiation in the primary immune response: dendritic cells isolated from adherent cell culture treated with IL-10 prime naive CD4+ T cells to secrete IL-4. Int Immunol. 1998 August; 10(8):1017-26). Human DC exposed to IL-10 induce a state of antigen-specific anergy in CD4+ T cells and CD8+ T cells by similarly converting DC into an immuoregulatory state (104). IL-10 inhibits IL-12 production and co-stimulatory molecule expression by DC, giving rise to regulatory DC (Kalinski P, Hilkens C M, Wierenga E A, Kapsenberg M L. T-cell priming by type-1 and type-2 polarized dendritic cells: the concept of a third signal. Immunol Today. 1999 December; 20(12):561-7).
DC could also be manipulated in situ to induce peripheral tolerance. For example Flt3L, a growth factor that expands DC, enhanced the induction of oral tolerance in vivo (Viney J L, Mowat A M, O'Malley J M, Williamson E, Fanger N A. Expanding dendritic cells in vivo enhances the induction of oral tolerance. J. Immunol. 1998 Jun. 15; 160(12):5815-25). By contrast, treatment with Flt-3L increased severity of experimental autoimmune thyroiditis due to enhanced Th1 responses, while GM-CSF either prevented or significantly suppressed disease development even at a late stage, due to enhanced Th2 responses (Vasu C, Dogan R N, Holterman M J, Prabhakar B S. Selective induction of dendritic cells using granulocyte macrophage-colony stimulating factor, but not fms-like tyrosine kinase receptor 3-ligand, activates thyroglobulin-specific CD4+/CD25+ T cells and suppresses experimental autoimmune thyroiditis. J. Immunol. 2003 Jun. 1; 170(11):5511-22).
Several procedures to induce tolerance have been developed using either DC modified as just described, or different routes of DC administration. For example, subcutaneous (sc) injection of antigen-pulsed splenic DC or epidermal Langerhans cells induces antigen-specific immunity, whereas intravenous (iv) injections of the same preparation result in tolerance (Morikawa Y, Furotani M, Kuribayashi K, Matsuura N, Kakudo K. The role of antigen-presenting cells in the regulation of delayed-type hypersensitivity. I. Spleen dendritic cells. Immunology. 1992 September; 77(1):81-7; Morikawa Y, Furotani M, Matsuura N, Kakudo K. The role of antigen-presenting cells in the regulation of delayed-type hypersensitivity. II. Epidermal Langerhans' cells and peritoneal exudate macrophages. Cell Immunol. 1993 November; 152(1):200-10). Specific strategies for autoimmune diseases might include promotion of regulatory T cell development using regulatory DC, or genetic engineering of DC to introduce molecules that have immunosuppressive functions, such as IL-10, TGFβ, Fas-ligand, ILT3 and ILT4. Evidence for the ability of DC to suppress autoimmune inflammatory disease so far comes from the application of DC to models of autoimmune disease, as detailed below. Syngeneic DC, with or without exposure to autoantigens have been shown to inhibit the development of autoimmune diseases of the neuromuscular system, such as experimental allergic encephalomyelitis (EAE), autoimmune endocrinopathies, such as type 1 diabetes and models of autoimmune arthritis, such as collagen-induced arthritis.
After exposure to TGFβ in vitro, splenic DC from healthy syngeneic donor rats could transfer suppression to recipients with EAE. In contrast, TGFβ-exposed DC from donor rats with EAE had no effect when transferred. DC were administered 5 days after immunization of Lewis rats with encephalitogenic myelin basic protein peptide 68-86 (MBP68-86) and complete Freund's adjuvant (CFA), during the incipient phase of EAE (Huang Y M, Yang J S, Xu L Y, Link H, Xiao B G. Autoantigen-pulsed dendritic cells induce tolerance to experimental allergic encephalomyelitis (EAE) in Lewis rats. Clin Exp Immunol. 2000 December; 122(3):437-44). Sc injection of immature, but not lipopolysaccharide (LPS)-treated, bone marrow (BM)-derived DC prior to immunization also prevented EAE (Xiao B G, Huang Y M, Yang J S, Xu L Y, Link H. Bone marrow-derived dendritic cells from experimental allergic encephalomyelitis induce immune tolerance to EAE in Lewis rats. Clin Exp Immunol. 2001 August; 125(2):300-9). TGFβ-modified DC similarly inhibited the development of clinical signs of experimental autoimmune myasthenia gravis (EAMG) in Lewis rats when given during the incipient phase of EAMG (Yarilin D, Duan R, Huang Y M, Xiao B G. Dendritic cells exposed in vitro to TGF-beta1 ameliorate experimental autoimmune myasthenia gravis. Clin Exp Immunol. 2002 February; 127(2):214-9).
In autoimmune disease of the eye, peptide-loaded immature DC inhibited the production of IFN-γ by uveitogenic T cells and therefore the induction of experimental autoimmune uveo-retinitis (EAU) in vivo (Jiang H R, Muckersie E, Robertson M, Forrester J V. Antigen-specific inhibition of experimental autoimmune uveoretinitis by bone marrow-derived immature dendritic cells. Invest Opthalmol V is Sci. 2003 April; 44(4):1598-607). Draining lymph node T cells secreted high levels of IL-10 and IL-15. In another model, transfer of inter-photoreceptor retinoid binding protein-pulsed TGFβ2-treated APC to inter-photoreceptor retinoid binding protein-immunized mice successfully suppressed the induction of experimental uveoretinitis in mice (Okamoto S, Kosiewicz M, Caspi R, Streilein J. ACAID as a potential therapy for establishmental autoimmune uveitis. In: Science E, editor. Advances in Ocular Immunology. Amsterdam; 1994).
Myelin antigen-pulsed splenocytes were shown to suppress EAE by selective induction of anergy in encephalitogenic T cells (Vandenbark A A, Celnik B, Vainiene M, Miller S D, Offner H. Myelin antigen-coupled splenocytes suppress experimental autoimmune encephalomyelitis in Lewis rats through a partially reversible anergy mechanism. J. Immunol. 1995 Dec. 15; 155(12):5861-7). Regulatory APC, generated by exposure to TGFb2 and MBP Ag, promoted development of CD8+ Treg that suppressed EAE (Faunce D E, Terajewicz A, Stein-Streilein J. Cutting edge: in vitro-generated tolerogenic APC induce CD8+ T regulatory cells that can suppress ongoing experimental autoimmune encephalomyelitis. J. Immunol. 2004 Feb. 15; 172(4):1991-5): These results provide evidence that DC can induce tolerance in experimental autoimmune diseases through effects on responding T cells. In alternative approach, EAE could be prevented by iv injection of splenic DC exposed ex vivo to MBP and CTLA-4-Ig fusion protein, presumably through ex vivo blockade of CD28-CD80 interactions (Khoury S J, Gallon L, Verburg R R, Chandraker A, Peach R, Linsley P S, et al. Ex vivo treatment of antigen-presenting cells with CTLA4Ig and encephalitogenic peptide prevents experimental autoimmune encephalomyelitis in the Lewis rat. J. Immunol. 1996 Oct. 15; 157(8):3700-5).
In a number of models, repetitive intravenous administration of so-called “semimature” DC, prepared in vitro by exposure to tumor necrosis factor TNF-α, induced Ag-specific protection. TNF-α-DC have been shown to express high levels of MHC and T cell co-stimulatory molecules, but unlike mature DC, they produce low levels of pro-inflammatory cytokines and are unable to secrete IL-12p70. These DC suppress EAE through generation of autoantigen-specific IL-10-secreting CD4+ T cells (Menges M, Rossner S, Voigtlander C, Schindler H, Kukutsch N A, Bogdan C, et al. Repetitive injections of dendritic cells matured with tumor necrosis factor alpha induce antigen-specific protection of mice from autoimmunity. J Exp Med. 2002 Jan. 7; 195(1):15-21), possibly as a result of the lack of expression of co-stimulatory “signal 3” (Thomas R. Signal 3 and its role in autoimmunity. Arthritis Res Ther. 2004; 6:26-7). Finally, DC exposed to TGF-β1 or IFN-γ suppressed the onset and relapses of EAE, in comparison with animals receiving untreated DC or saline injections (Xiao B G, Wu X C, Yang J S, Xu L Y, Liu X, Huang Y M, et al. Therapeutic potential of IFN-gamma-modified dendritic cells in acute and chronic experimental allergic encephalomyelitis. Int Immunol. 2004 January; 16(1):13-22).
In the NOD mouse model of diabetes, transfer of DC treated with IFN-γ also induced long-lasting protection against type 1 diabetes mellitus (Shinomiya M, Fazle Akbar S M, Shinomiya H, Onji M. Transfer of dendritic cells (DC) ex vivo stimulated with interferon-gamma (IFN-gamma) down-modulates autoimmune diabetes in non-obese diabetic (NOD) mice. Clin Exp Immunol. 1999 July; 117(1):38-43). Transfer of pancreatic lymph node DC also suppressed the development of diabetes by the induction of regulatory cells in NOD mice (Clare-Salzler M J, Brooks J, Chai A, Van Herle K, Anderson C. Prevention of diabetes in nonobese diabetic mice by dendritic cell transfer. J Clin Invest. 1992 September; 90(3):741-8). In other experiments, a single iv injection of syngeneic splenic DC from euglycemic NOD mice exposed to human IgG protected mice from diabetes. Supernatants of islets from these mice contained increased levels of IL-4 and IL-10 and diminished levels of IFN-γ compared with diabetic controls, suggesting a favorable effect of type 2 cytokines on disease (Papaccio G, Nicoletti F, Pisanti F A, Bendtzen K, Galdieri M. Prevention of spontaneous autoimmune diabetes in NOD mice by transferring in vitro antigen-pulsed syngeneic dendritic cells. Endocrinology. 2000 April; 141 (4): 1500-5).
Mature BM-derived DC could also prevent diabetes development in NOD mice, an effect ascribed to the generation of CD25+CD4+ regulatory T cells, secreting Th2 cytokines (Feili-Hariri M, Dong X, Alber S M, Watkins S C, Salter R D, Morel P A. Immunotherapy of NOD mice with bone marrow-derived dendritic cells. Diabetes. 1999 December; 48(12):2300-8). BM-derived DC generated in the presence of NF-κB inhibitory oligo-dinucleotides or the soluble NF-κB inhibitor Bay11-7082 could also prevent diabetes (Ma L, Qian S, Liang X, Wang L, Woodward J E, Giannoukakis N, et al. Prevention of diabetes in NOD mice by administration of dendritic cells deficient in nuclear transcription factor-kappaB activity. Diabetes. 2003 August; 52(8):1976-85). However, no studies have demonstrated that transferred DC can ameliorate established type 1 diabetes in NOD mice.
Experimental autoimmune thyroiditis (EAT), a murine model of Hashimoto's thyroiditis in humans, can be induced upon challenge of susceptible animals with thyroglobulin and adjuvant (Charreire J. Immune mechanisms in autoimmune thyroiditis. Adv Immunol. 1989; 46:263-334). This disease is mediated by CD4+ T cells and is characterized by lymphocytic infiltration of the thyroid gland (Weetman A P, McGregor A M. Autoimmune thyroid disease: further developments in our understanding. Endocr Rev. 1994 December; 15(6):788-830). DC exposed to TNFα and Ag induced Ag-specific CD4+CD25+ T cells with the ability to inhibit development of EAT, confirming results previously published in a model of EAE (Verginis P, Li H S, Carayanniotis G. Tolerogenic semimature dendritic cells suppress experimental autoimmune thyroiditis by activation of thyroglobulin-specific CD4+CD25+ T cells. J. Immunol. 2005 Jun. 1; 174(11):7433-9).
Several studies in experimental arthritis have evaluated the therapeutic effect of DC transduced with various immunomodulatory genes. Transduction of DC with TNF-related apoptosis-induced ligand (TRAIL) was evaluated in mice with collagen-induced arthritis (CIA). TRAIL expression was controlled by a doxycycline-inducible tetracycline response element. Transfected DC were capable of inducing apoptosis of arthritogenic T cells (Liu Z, Xu X, Hsu H C, Tousson A, Yang P A, Wu Q, et al. CII-DC-AdTRAIL cell gene therapy inhibits infiltration of CII-reactive T cells and CII-induced arthritis. J Clin Invest. 2003 November; 112(9):1332-41). Genetic modification of primary DC to express Fas-L eliminated or reduced the number of antigen-specific T cells responsible for the progression of CIA (Kim S H, Kim S, Oligino T J, Robbins P D. Effective treatment of established mouse collagen-induced arthritis by systemic administration of dendritic cells genetically modified to express FasL. Mol. Ther. 2002 November; 6(5):584-90). Moreover, DC transfected with Fas-L could induce antigen-specific tolerance after exposure to a peptide to which they had previously been sensitized. This observation provides evidence that it may also be possible to delete autoreactive T cells from the repertoire using modified DC (Matsue H, Matsue K, Walters M, Okumura K, Yagita H, Takashima A. Induction of antigen-specific immunosuppression by CD95L cDNA-transfected ‘killer’ dendritic cells. Nat. Med. 1999 August; 5(8):930-7).
Adoptive transfer of immature DC expressing IL-4 after adenoviral infection, into mice with established CIA suppressed disease for up to 4 weeks (Kim S H, Kim S, Evans C H, Ghivizzani S C, Oligino T, Robbins P D. Effective treatment of established murine collagen-induced arthritis by systemic administration of dendritic cells genetically modified to express IL-4. J. Immunol. 2001 Mar. 1; 166(5):3499-505). Similarly, IL-4-transduced bone marrow derived DC adoptively transferred before disease onset reduced the incidence and severity of murine CIA, whereas IL-4 delivery by retrovirally transduced T cells and NIH 3T3 cells had no effect (Morita Y, Yang J, Gupta R, Shimizu K, Shelden E A, Endres J, et al. Dendritic cells genetically engineered to express IL-4 inhibit murine collagen-induced arthritis. J Clin Invest. 2001 May; 107(10):1275-84). Whereas each of these approaches suppressed Th1-mediated T cell and antibody responses, they typically did not deviate the immune response towards a Th2 type or regulatory response. By contrast, DC generated in the presence of vasoactive intestinal peptide (VIP) were able to suppress CIA in an IL-10 dependent fashion (Chorny A, Gonzalez-Rey E, Fernandez-Martin A, Ganea D, Delgado M. Vasoactive intestinal peptide induces regulatory dendritic cells that can prevent acute graft-versus-host disease while maintain graft-versus-tumor. Blood. 2006 Jan. 17). TNF-DC also suppressed CIA, when delivered i.v. in high doses, in a partially IL-10 dependent manner (Verginis P, Li H S, Carayanniotis G. Tolerogenic semimature dendritic cells suppress experimental autoimmune thyroiditis by activation of thyroglobulin-specific CD4+CD25+ T cells. J. Immunol. 2005 Jun. 1; 174(11):7433-9). Both TNF-DC and VIP-DC stimulate peripheral conversion of CD4+CD25+ regulatory T cells and Tr1 type Treg. VIP has been shown to reduce DC NF-κB activation and CD40 expression (Chorny A, Gonzalez-Rey E, Fernandez-Martin A, Ganea D, Delgado M. Vasoactive intestinal peptide induces regulatory dendritic cells that can prevent acute graft-versus-host disease while maintain graft-versus-tumor. Blood. 2006 Jan. 17).
DC immunotherapy has been introduced in the clinic, and has proven to be feasible, non-toxic and effective in some patients with cancer, particularly if the DC have been appropriately activated (Banchereau J, Palucka A K, Dhodapkar M, Burkeholder S, Taquet N, Rolland A, et al. Immune and clinical responses in patients with metastatic melanoma to CD34(+) progenitor-derived dendritic cell vaccine. Cancer Res. 2001 Sep. 1; 61(17):6451-8; Nestle F O, Banchereau J, Hart D. Dendritic cells: On the move from bench to bedside. Nat. Med. 2001 July; 7(7):761-5; Dhodapkar M V, Krasovsky J, Steinman R M, Bhardwaj N. Mature dendritic cells boost functionally superior CD8(+) T-cell in humans without foreign helper epitopes. J Clin Invest. 2000 March; 105(6):R9-R14). In vivo activation and targeting of DC, as well as exploitation of DC to suppress autoimmunity, will expand the application of DC to a wide variety of immune-mediated diseases. However, a number of technical questions also need to be addressed in autoimmune immunotherapy, including the frequency and route of administration, the subset and number of DC to be used, and the concentration and duration of cytokine treatment. For example, while a single iv or sc dose of 0.5×106 DC treated with an NF-κB inhibitor was sufficient to suppress priming or antigen-induced arthritis, TNF-treated DC must be given repeatedly iv in high doses.
Data relating to human DC are scarce, but certain studies have reported encouraging results. Using a human in vitro model system, immature DC exposed to allospecific CD8+CD28− T suppressor cells or CD4+CD25+ Treg exhibited increased surface expression of the inhibitory molecules ILT3 and 4 (Chang C C, Ciubotariu R, Manavalan J S, Yuan J, Colovai A I, Piazza F, et al. Tolerization of dendritic cells by T(S) cells: the crucial role of inhibitory receptors ILT3 and ILT4. Nat. Immunol. 2002 March; 3(3):237-43). These human regulatory DC induced reversible anergy in unprimed or primed T helper cells, promoting the conversion of alloreactive CD4+ T cells to Treg. Human blood CD4+CD123+CD11c− precursor DC can be generated when cultured in the presence of IL-3 (Grouard G, Rissoan M C, Filgueira L, Durand I, Banchereau J, Liu Y J. The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand. J Exp Med. 1997 Mar. 17; 185(6):1101-11; Rissoan M C, Soumelis V, Kadowaki N, Grouard G, Briere F, de Waal Malefyt R, et al. Reciprocal control of T helper cell and dendritic cell differentiation. Science. 1999 Feb. 19; 283(5405):1183-6; Arpinati M, Green C L, Heimfeld S, Heuser J E, Anasetti C. Granulocyte-colony stimulating factor mobilizes T helper 2-inducing dendritic cells. Blood. 2000 Apr. 15; 95(8):2484-90). After in vitro activation by TNF-α, these DC promoted production of IL-4 and IL-10 by T cells (Rissoan M C, Soumelis V, Kadowaki N, Grouard G, Briere F, de Waal Malefyt R, et al. Reciprocal control of T helper cell and dendritic cell differentiation. Science. 1999 Feb. 19; 283(5405):1183-6). Such DC have potential for the treatment of autoimmune diseases and acute graft-versus-host disease (Liu Y J, Blom B. Introduction: TH2-inducing DC2 for immunotherapy. Blood. 2000 Apr. 15; 95(8):2482-3).
PB monocyte-derived DC, exposed to IFN-β secrete high levels of IL-10 but low levels of IL-12, and suppress IFN-γ production by mononuclear cells (Huang Y M, Hussien Y, Yarilin D, Xiao B G, Liu Y J, Link H. Interferon-beta induces the development of type 2 dendritic cells. Cytokine. 2001 Mar. 7; 13(5):264-71). DC from MS patients treated with IFN-β in vivo produced less IFN-γ and TNF-α than DC from control patients (Huang Y M, Xiao B G, Ozenci V, Kouwenhoven M, Teleshova N, Fredrikson S, et al. Multiple sclerosis is associated with high levels of circulating dendritic cells secreting pro-inflammatory cytokines. J. Neuroimmunol. 1999 Sep. 1; 99(1):82-90). These findings suggest that exposure of DC to IFN-β and IL-10 may curtail the production of pro-inflammatory cytokines, and after re-infusion, such DC may represent a promising direction for therapy of MS. Signaling through NF-κB was also shown to determine the capacity of DC to stimulate T cell proliferation in vitro, in that CD40− human monocyte-derived DC generated in the presence of an NF-κB inhibitor, signal little T cell proliferation or IFN-γ production (Thompson A G, O'Sullivan B J, Beamish H, Thomas R. T cells signaled by NF-kappa B-dendritic cells are sensitized not anergic to subsequent activation. J. Immunol. 2004 Aug. 1; 173(3):1671-80).
In a human study of two healthy volunteers, in vivo responses to recall antigens were suppressed when normal volunteers were injected with antigen-exposed immature DC (Dhodapkar M V, Steinman R M, Krasovsky J, Munz C, Bhardwaj N. Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med. 2001 Jan. 15; 193(2):233-8). This effect was linked to the generation of regulatory type CD4+ and CD8+ T cells and the production of IL-10, and is in marked contrast to the active immunity that can be achieved with mature DC. This small study is the only clinical evidence to date illustrating the potential of immature DC as a tool for immunosuppression. However, it is not yet clear whether this potential will translate into patients with immune system defects that have led to the development of spontaneous autoimmune disease.
Patients with systemic lupus erythematosus (SLE) have been shown to display major alterations in DC homeostasis in that plasmacytoid DC are reduced in blood and IFNα-activated monocytes from these patients are effective APC in vitro (Blanco P, Palucka A K, Gill M, Pascual V, Banchereau J. Induction of dendritic cell differentiation by IFN-alpha in systemic lupus erythematosus. Science. 2001; 294(5546):1540-3). It was speculated that monocyte-derived DC might efficiently capture apoptotic cells and nucleosomes, present in SLE patients' blood and tissues (Amoura Z, Piette J C, Chabre H, Cacoub P, Papo T, Wechsler B, et al. Circulating plasma levels of nucleosomes in patients with systemic lupus erythematosus: correlation with serum antinucleosome antibody titers and absence of clear association with disease activity. Arthritis Rheum. 1997; 40(12):2217-25). In view of the high levels of IFNα in serum, and its detrimental effects in SLE, IFNα is being developed as a potential target for therapeutic intervention in SLE (Vallin H, Blomberg S, Alm G V, Cederblad B, Ronnblom L. Patients with systemic lupus erythematosus (SLE) have a circulating inducer of interferon-alpha (IFN-alpha) production acting on leucocytes resembling immature dendritic cells. Clin Exp Immunol. 1999; 115(1):196-202). IFNα activates not only myeloid cells, including monocytes and myeloid DC, but also plasmacytoid DC themselves, which are enriched in the inflammatory site in SLE skin lesions (Farkas L, Beiske K, Lund-Johansen F, Brandtzaeg P, Jahnsen F L. Plasmacytoid Dendritic Cells (Natural Interferon-alpha/beta-Producing Cells) Accumulate in Cutaneous Lupus Erythematosus Lesions. Am J. Pathol. 2001; 159(1):237-43). Of interest, the RNA components of the Ro 60 and Sm/RNP small ribonucleoprotein autoantigens have recently been shown to act as endogenous adjuvants which stimulate plasmacytoid DC (PDC) maturation and type I IFN production (Kelly K M, Zhuang H, Nacionales D C, Scumpia P O, Lyons R, Akaogi J, et al. “Endogenous adjuvant” activity of the RNA components of lupus autoantigens Sm/RNP and Ro 60. Arthritis Rheum. 2006 Apr. 27; 54(5):1557-67; Savarese E, Chae O W, Trowitzsch S, Weber G, Kastner B, Akira S, et al. U1 small nuclear ribonucleoprotein immune complexes induce type I interferon in plasmacytoid dendritic cells through TLR7. Blood. 2006 Apr. 15; 107(8):3229-34; Vollmer J, Tluk S, Schmitz C, Hamm S, Jurk M, Forsbach A, et al. Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8. J Exp Med. 2005 Dec. 5; 202(11):1575-85). Type I IFN production by PDC can also be triggered in cutaneous LE by UV-light, which stimulates local production of chemokines for T cells and PDC.
Additionally, several investigators have postulated in vivo administration of soluble inhibitors of the NF-κB pathway either by themselves or in combination with soluble antigens to elicit tolerogenic DC for the treatment of autoimmune disease, allergies and graft versus host disease. Illustrative references disclosing this strategy include: U.S. Pat. No. 7,078,027; U.S. Pat. App. Pub. Nos. 2005/032725, 2004/072228, 2004/166095, 2004/166099, 2005/208036, 2004/258688, 2004/265912, 2005/0220854, 2005/0036993 and 2003/0153518; International Publications WO 99/29865, WO 00/61132, WO 03/000199, WO 2004/084927 and WO 2004/084942; and European Pat. App. No. 1 462 111. However, there is no clinical evidence to the knowledge of the present inventors that supports the usefulness of this strategy.
The present invention is predicated in part on the surprising discovery that co-administration of an NF-κB inhibitor and an antigen in vivo when both are in soluble form or when one is soluble and the other is liposome encapsulated, is ineffective in producing a tolerogenic response to the antigen. However, the present inventors have found that strong tolerogenic responses are generated in vivo by administering particles (e.g., liposomes) comprising both an NF-κB inhibitor and an antigen. This discovery has been reduced to practice in the form of particulate, immunomodulating compositions and methods of treating or preventing undesirable or deleterious immune responses, as described hereafter.